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US6723322B1 - Angiogenic Onchocerca volvulus proteins and uses thereof - Google Patents

Angiogenic Onchocerca volvulus proteins and uses thereof Download PDF

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US6723322B1
US6723322B1 US09/541,759 US54175900A US6723322B1 US 6723322 B1 US6723322 B1 US 6723322B1 US 54175900 A US54175900 A US 54175900A US 6723322 B1 US6723322 B1 US 6723322B1
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asp
tissue
lys
gly
leu
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Sara Lustigman
Eric Pearlman
Thomas R. Unnasch
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Case Western Reserve University
UAB Research Foundation
New York Blood Center Inc
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Case Western Reserve University
UAB Research Foundation
New York Blood Center Inc
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Priority to AT01920807T priority patent/ATE372128T1/de
Priority to CA2403985A priority patent/CA2403985C/fr
Priority to PCT/US2001/009798 priority patent/WO2001074385A1/fr
Priority to EP01920807A priority patent/EP1267917B1/fr
Priority to AU4782501A priority patent/AU4782501A/xx
Priority to DE60130326T priority patent/DE60130326T2/de
Priority to AU2001247825A priority patent/AU2001247825B2/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Angiogenesis refers to the process by which new capillary blood vessels are formed from existing microvessels, resulting in the development of a blood supply to a given area of tissue [23, 25]. It is one of the most pervasive and fundamentally essential biological processes encountered in mammalian organizations. In the healthy, adult human body, angiogenesis is a normal and important function that is critical in a variety of physiological settings, including chronic inflammation, embryonic development, reproduction, and wound healing [22, 29]. For example, angiogenesis occurs in the female reproductive system, in response to ovulation or gestation, and in the normal hair cycle [28]. Nevertheless, apart from the processes of wound healing and inflammation, angiogenesis virtually never occurs physiologically in adult tissues, except in the ovary, the endometrium, and the placenta [27].
  • angiogenesis is also central to a number of pathological processes, including: abnormalities of wound healing in diseases such as diabetes and duodenal ulceration; chronic inflammatory disorders, such as rheumatoid arthritis, psoriasis, and periodontitis; dermatological conditions such as cutaneous malignancy, decubitus ulcers, hemangiomas, Kaposi's sarcoma, psoriasis, pyogenic granulomas, and warts; diseases of the eye, particularly diabetic retinopathy; and growth of solid tumors, both benign and malignant [22, 23, 25, 26].
  • the consequence of abnormal angiogenesis is either excessive or insufficient blood vessel growth. Ulcers, strokes, and heart attacks, for example, can result from the absence of angiogenesis normally required for natural healing, while excessive blood vessel proliferation may favor arthritis, blindness, and tumor growth and dissemination [29 ].
  • the angiogenic process is tightly regulated—in both time and space—by a variety of endogenous angiogenic and angiostatic factors. It is propelled by a mixture of growth factors and pro-angiogenic cytokines, and is moderated by a collection of inhibitors of neovascularization which interfere with steps in the angiogenic process [22, 30].
  • angiogenesis capillary sprouts are formed in response to pro-angiogenic factors. The sprouts then grow and develop, driven by endothelial cell migration and proliferation, and organize themselves into a ordendritic structure [24].
  • Angiogenic and anti-angiogenic molecules control the formation of new vessels via different mechanisms.
  • Processes which are necessary for new vessel formation, and which are regulated by angiogenic and anti-angiogenic molecules, include the migration and proliferation of endothelial cells from the microvasculature, the controlled expression of proteolytic enzymes, the breakdown and reassembly of extracellular matrix, and the morphogenic process of endothelial tube formation.
  • some angiogenesis-dependent diseases can be controlled or modulated via induction or inhibition of new vessel formation [23].
  • angiogenesis-dependent diseases or conditions including cancer, diabetic retinopathy, inflammatory diseases, ischemic heart disease, myocardial infarction, peripheral vascular disease, and wound healing.
  • Onchocerciasis occurs primarily as a result of a host inflammatory response to infection with the filarial nematode Onchocerca volvulus ( O volvulus ). Transmitted by the bites of blackflies from the family Simuliidae, which breed in swiftly flowing streams, the parasite invades the skin, subcutaneous tissues, and other tissues, producing fibrous nodules.
  • the host inflammatory response to infection with O volvulus may manifest in chronic skin disease and eye lesions. In the cornea, for example, this response produces neovascularization—the seminal event in the pathologic response process—followed by corneal opacification.
  • Ocular onchocerciasis is characterized by lesions of the anterior eye, including punctate keratitis, deformation of the pupil, and an ingrowth of fibrovascular scar tissue that may result in blindness.
  • onchocerciasis is the second leading cause of infectious blindness in the world. Of the 18 million people who are believed to be infected with onchocerciasis, approximately 270,000 are blind, and a further 500,000 are visually impaired [1-4, 31].
  • O. volvulus A library of expressed sequence tags of O. volvulus has recently been developed by the Filarial Genome Project, and numerous cDNAs have been cloned [5]. From this library, a number of O. volvulus proteins, including Ov20 [32], OvPDI [11], and Ovzf [33], have been characterized. However, the relationship between proteins of O. volvulus and the host inflammatory response in ocular onchocerciasis—particularly in the induction of corneal neovascularization—has not been fully delineated.
  • the present invention is based on the discovery that certain members of the Ov-ASP protein family are involved in the pathologic process of corneal neovascularization in animals infected with O. volvulus .
  • This discovery which indicates a pro-angiogenic role for Ov-ASP proteins, will have implications for wound healing and for the treatment of diseases, such as ischemia, where the enhancement or promotion of angiogenesis is desirable.
  • this discovery permits screening for anti-Ov-ASP factors which inhibit or reduce the angiogenic activity of Ov-ASP. This finding will have implications in the treatment of ocular onchocerciasis.
  • the present invention provides a method for inducing angiogenesis in a tissue by contacting the tissue with an amount of Ov-ASP effective to induce angiogenesis in the tissue.
  • the present invention further provides a method for screening for an anti-Ov-ASP factor, by contacting a factor of interest with Ov-ASP, and then assessing the ability of the factor to inhibit angiogenic activity of Ov-ASP.
  • the present invention provides a method for inhibiting angiogenesis in a subject.
  • FIG. 1 presents a comparison between members of the Ov-ASP family and other members of the Tpx and CRISP families of protein. Sequences were aligned using the MACAW program, as described in Materials and Methods. Grey shading highlights areas with high mean similarity scores, while black shading highlights identical amino acids. Italics highlight the putative signal sequences identified, as described in Material and Methods. Asterisks highlight the conserved cysteine residues discussed in the text.
  • Ov-ASP 1 SEQ ID NO:1
  • Ov-ASP-2 SEQ ID NO:2
  • Ov-ASP-3 SEQ ID NO:3
  • Bm-ASP Brugia malayi ASP homologue
  • Ac-ASP SEQ ID NO:5
  • FIG. 2 depicts a phylogenetic analysis of members of the Ov-ASP family.
  • FIG. 3 provides a genomic Southern blot analysis of members of the Ov-ASP family.
  • a genomic Southern blot was prepared with O. vovulus and human restriction-digested DNA was probed with a purified insert encoding the full-length open reading frame of Ov-asp-2, as described in Materials and Methods.
  • Lane 1 O. vovulus genomic DNA digested with BamHl
  • Lane 2 O. volvulus genomic DNA digested with HindIII
  • Lane 3 human genomic DNA digested with BamH1
  • Lane 4 human genomic DNA digested with HindIII
  • FIG. 4 illustrates stage-specific PCR showing expression profiles of the three O. volvulus asp genes in different stages of the life cycle. Amplifications were carried out using gene-specific primers on cDNA libraries prepared from microfilariae (mf), second-stage larvae (L2), third-stage larvae (L3), molting larvae (mL3), fourth-stage larvae (L4), and adult male and female.
  • the O. Volvulus gene encoding glyceraldehyde 3-phosphate dehydrogenase (Ov-gpd-1) was included as an internal control.
  • FIG. 5 depicts an angiogenic response elicited by Ov-ASP-2.
  • Panel A Thirty ⁇ g of Ov-ASP-2/MBP was injected into the corneal stroma of BALB/c mice. Growth of blood vessels was monitored by length (vessel length) and by circumferential area (clock hours) from the injection site over time, as described in Materials and Methods. Data are means ⁇ S.D. of 5 mice per group. This experiment was repeated 5 times with similar results.
  • Panel B Photomicrograph of BALB/c mouse, 7 days after injection of Ov-ASP-2/MBP. The asterisk indicates the site of injection.
  • FIG. 6 demonstrates the specificity of the angiogenic response elicited by Ov-ASP/MBP fusion proteins.
  • BALB/c mice were injected in the corneal stroma with Ov-ASP-1/MBP, Ov-ASP-2/MBP, Ov-PDI/MBP, or MBP alone. Results shown are from day 3 after injection, and represent means ⁇ S.D. of 5 mice per group. This experiment was repeated 5 times with similar results.
  • FIG. 7 depicts the histology of mouse cornea, 7 days after injection of Ov-ASP-2/MBP. Numerous blood vessels are present in the corneal stroma (large arrows), but only occasional mononuclear cells (small arrows) are present. The micrograph is representative of 5 animals per group, and 5 replicated experiments.
  • the present invention is directed to a method for inducing angiogenesis in a tissue, comprising contacting the tissue with an amount of Ov-ASP effective to induce angiogenesis in the tissue.
  • Ov-ASP is used to refer to members of the Ov-ASP protein family and analogues thereof, as well as homologues from other species which induce angiogenesis.
  • Members of the Ov-ASP protein family include Ov-ASP-1, Ov-ASP-2, and Ov-ASP-3.
  • Analogues of Ov-ASP include, for example, a functional variant of wild-type Ov-ASP protein which has Ov-ASP biological activity, as well as a fragment of Ov-ASP having Ov-ASP biological activity.
  • Ov-ASP biological activity refers to Ov-ASP activity which induces angiogenesis.
  • the contacting of tissue with Ov-ASP may be effected by introducing a nucleic acid encoding Ov-ASP in a manner permitting expression of the Ov-ASP protein, or by introducing the Ov-ASP protein itself.
  • the method of the present invention may also be used to induce angiogenesis in vivo or in vitro.
  • Tissue may be contacted with Ov-ASP by introducing to the tissue a nucleic acid encoding Ov-ASP, in a manner permitting expression of Ov-ASP.
  • Nucleic acid encoding Ov-ASP may be genomic DNA or cDNA.
  • the nucleic acid may be introduced using conventional procedures known in the art, including, without limitation, electroporation, DEAE Dextran transfection, calcium phosphate transfection, monocationic liposome fusion, polycationic liposome fusion, protoplast fusion, creation of an in vivo electrical field, DNA-coated microprojectile bombardment, injection with recombinant replication-defective viruses, homologous recombination, gene therapy, viral vectors, naked DNA transfer, or a combination thereof. It is to be appreciated by one skilled in the art that any of the above methods of DNA transfer may be combined.
  • a nucleic acid encoding Ov-ASP may be introduced to a tissue or subject using gene therapy, e.g., by introducing a recombinant vector containing a nucleic acid sequence encoding Ov-ASP.
  • the nucleic acid sequence may be, for example, genomic DNA or cDNA.
  • the recombinant vector containing nucleic acid encoding Ov-ASP may be administered to a subject using any number of procedures known to one skilled in the art, including, without limitation, electroporation, DEAE Dextran transfection, calcium phosphate transfection, monocationic liposome fusion, polycationic liposome fusion, protoplast fusion, creation of an in vivo electrical field, DNA-coated microprojectile bombardment, injection with recombinant replication-defective viruses, homologous recombination, gene therapy, viral vectors, naked DNA transfer, or a combination thereof. It is to be appreciated by one skilled in the art that any of the above methods of DNA transfer may be combined.
  • the recombinant vector may comprise a nucleic acid of, or corresponding to at least a portion of, the genome of a virus, where this portion is capable of directing the expression of a nucleic sequence encoding Ov-ASP, operably linked to the viral nucleic acid and capable of being expressed as a functional gene product in the tissue or subject.
  • Recombinant viral vectors suitable for gene therapy may be derived from a variety of viral nucleic acids known to one skilled in the art, including, without limitation, the genomes of retrovirus, HSV, adenovirus, adeno-associated virus, Semiliki Forest virus, cytomegalovirus, vaccinia virus, and DNA and RNA viruses.
  • the recombinant vectors may also contain a nucleotide sequence encoding suitable regulatory elements so as to effect expression of the vector construct in a suitable host cell.
  • expression refers to the ability of the vector to transcribe the inserted DNA sequence into mRNA, so that synthesis of the protein encoded by the inserted nucleic acid can occur.
  • enhancers and promoters are suitable for use in the constructs of the invention, and that the constructs will contain the necessary start, termination, and control sequences for proper transcription and processing of the nucleic acid sequence encoding Ov-ASP when the recombinant vector construct is introduced into a subject.
  • Vectors suitable for the expression of the nucleic sequence encoding Ov-ASP are well known to one skilled in the art.
  • Suitable promoters include, but are not limited to, constitutive promoters, tissue specific promoters, and inducible promoters. Expression of the nucleic acid sequence encoding Ov-ASP may be controlled and affected by the particular vector into which the nucleic acid sequence has been introduced. Some eukaryotic vectors have been engineered so that they are capable of expressing inserted nucleic acids to high levels within the target cell. Such vectors utilize one of a number of powerful promoters to direct the high level of expression. Eukaryotic vectors use promoter-enhancer sequences of viral genes, especially those of tumor viruses. A particular embodiment of the invention provides for regulation of expression of the nucleic acid sequence encoding Ov-ASP using inducible promoters.
  • Non-limiting examples of inducible promoters include, but are not limited to, metallothionine promoters and mouse mammary tumor virus promoters.
  • expression of the nucleic acid sequence encoding Ov-ASP would be induced in tissue of a subject by the addition of a specific compound at a certain point in the growth cycle of the cells of the subject.
  • promoters and enhancers effective for use in the recombinant vectors include, but are not limited to, CMV (cytomegalovirus), SV40 (simian virus 40), HSV (herpes simplex virus), EBV (epstein-barr virus), retroviral, adenoviral promoters and enhancers, and tumor cell specific promoters and enhancers.
  • CMV cytomegalovirus
  • SV40 simian virus 40
  • HSV herpes simplex virus
  • EBV epstein-barr virus
  • retroviral adenoviral promoters and enhancers
  • adenoviral promoters and enhancers include, but are not limited to, CMV (cytomegalovirus), SV40 (simian virus 40), HSV (herpes simplex virus), EBV (epstein-barr virus), retroviral, adenoviral promoters and enhancers, and tumor cell specific promoters and enhancers.
  • a recombinant vector containing nucleic acid encoding Ov-ASP may be combined with a sterile aqueous solution which is preferably isotonic with the blood of the recipient.
  • a sterile aqueous solution which is preferably isotonic with the blood of the recipient.
  • Such formulations may be prepared by suspending the recombinant vector in water containing physiologically compatible substances such as sodium chloride, glycine, and the like, and having buffered pH compatible with physiological conditions to produce an aqueous solution, and rendering such solution sterile.
  • the recombinant vector is combined with a 20-25% sucrose in saline solution in preparation for introduction into a subject.
  • nucleic acid encoding Ov-ASP may be introduced into suitable cells in vitro using conventional procedures.
  • Cells expressing Ov-ASP may then be introduced into tissue of a subject in order to induce angiogenesis.
  • the cells are preferably removed from the subject, subjected to DNA techniques to incorporate the nucleic acid encoding Ov-ASP, and then reintroduced into the subject.
  • Nucleic acid encoding Ov-ASP or nucleic acid encoding Ov-ASP contained in a vector, is introduced to tissue of a subject in an amount effective to induce angiogenesis in the tissue.
  • the exact dosage will depend on such factors as the purpose of administration, the mode of administration, and the efficacy of the composition, as well as the individual pharmacokinetic parameters of the subject.
  • Such therapies may be administered as often as is necessary, and for the period of time determined necessary by one skilled in the art.
  • tissue may also be contacted with Ov-ASP by introducing to the tissue an Ov-ASP protein.
  • the Ov-ASP protein may be produced synthetically or recombinantly, or may be isolated from native cells; however, it is preferably produced recombinantly, using cDNA encoding Ov-ASP (Ov-asp-1: GenBank accession number AF020586; Ov-asp-2: GenBank accession number H39490; and Ov-asp-3: GenBank accession number AA917267), along with conventional techniques.
  • Ov-ASP-1, Ov-ASP-2, and Ov-ASP-3 proteins have the amino acid sequences set forth in FIG. 1 .
  • the Ov-ASP protein may be introduced to tissue of a subject in vivo by known techniques used for the introduction of proteins, including, for example, injection, transfusion, or topical application. Injection or transfusion of Ov-ASP may be effected, for example, intradermally, intramuscularly, intraperitoneally, intravenously, or subcutaneously. When tissue in a subject is localized to a particular portion of the subject's body, it may be desirable to introduce the protein directly to the tissue by site-directed injection to a specific organ, or by some other means (e.g., by introducing Ov-ASP into the blood or another body fluid).
  • the amount of Ov-ASP protein to be used is an amount effective to induce angiogenesis, and may be readily determined by the skilled artisan.
  • Ov-ASP The ability of Ov-ASP to induce neovascularization renders Ov-ASP particularly useful for treating subjects suffering from diseases or conditions associated with a need for angiogenesis.
  • the subject is preferably a mammal (e.g., humans, domestic animals, and commercial animals), and is most preferably a human. It is believed that, by inducing angiogenesis, Ov-ASP will be useful for the treatment of diseases or conditions where the enhancement or promotion-of angiogenesis is desirable. It is further believed that Ov-ASP would be effective either alone or in combination with therapeutic agents (e.g., chemotherapeutic agents or antiviral agents) or angiogenic factors (e.g., agents which induce, enhance, or promote angiogenesis) used in the treatment of these diseases or conditions.
  • therapeutic agents e.g., chemotherapeutic agents or antiviral agents
  • angiogenic factors e.g., agents which induce, enhance, or promote angiogenesis
  • Ov-ASP induces angiogenesis.
  • Ov-ASP may be used accelerate or enhance various biological processes associated with angiogenesis.
  • Ov-ASP may be administered to enhance wound healing and organ transplantation, including the transplantation of artificial organs. Therefore, this invention is directed to a method of enhancing wound healing in a subject by administering Ov-ASP, and to a method of enhancing organ transplantation in a subject by administering Ov-ASP.
  • Ov-ASP may be used to accelerate endothelial cell coverage of vascular grafts in order to prevent graft failure due to reocclusion. It may also be administered to enhance skin grafting.
  • the present invention also provides a method for screening for an anti-Ov-ASP factor.
  • An “anti-Ov-ASP factor” is a natural or synthetic agent which antagonizes Ov-ASP by reducing or inhibiting Ov-ASP biological activity (i.e., Ov-ASP angiogenic activity). It is also within the confines of the present invention that an anti-Ov-ASP factor is a natural or synthetic agent which antagonizes other angiogenic factors (e.g., agents which induce, enhance, or promote angiogenesis), including analogues and homologues of Ov-ASP, by reducing or inhibiting their angiogenic activity.
  • angiogenic factors e.g., agents which induce, enhance, or promote angiogenesis
  • the antibody of the present invention may be polyclonal or monoclonal, and may be produced by techniques well known to those skilled in the art.
  • Polyclonal antibody for example, may be produced by immunizing a mouse, rabbit, or rat with purified Ov-ASP.
  • Monoclonal antibody may then be produced by removing the spleen from the immunized mouse, and fusing the spleen cells with myeloma cells to form a hybridoma which, when grown in culture, will produce a monoclonal antibody.
  • the antibody of the present invention also includes a humanized antibody, made in accordance with procedures known in the art.
  • an anti-Ov-ASP factor may be screened using in vitro assays.
  • an anti-Ov-ASP factor may be screened by contacting a factor of interest with Ov-ASP, then assessing the ability of the factor to inhibit angiogenic activity of Ov-ASP.
  • a corneal assay may be used to screen for an anti-Ov-ASP factor.
  • An agent of interest may be brought into contact with corneal tissue or cells containing Ov-ASP, then the corneal tissue or cells may be assessed to determine if the agent of interest reduces or inhibits angiogenesis or neovascularization in the corneal tissue or cells.
  • an in vitro assay using human vascular endothelial cells or human umbilical vein endothelial cells may be utilized to screen for an anti-Ov-ASP factor.
  • An anti-Ov-ASP factor identified by the above-described screening method is also provided by the present invention.
  • the present invention further provides a method for inhibiting angiogenesis in a subject.
  • the method of the present invention comprises administering to a subject an amount of anti-Ov-ASP factor effective to inhibit angiogenesis in the subject.
  • the amount of anti-Ov-ASP factor required to inhibit angiogenesis may be readily determined by one skilled in the art.
  • the present method for inhibiting angiogenesis in a subject would be useful for treating a subject having a disease or condition where the inhibition of angiogenesis would be desirable, including, without limitation, arthritis, corneal disease, diabetic retinopathy, pyogenic granulomas, hypertrophic scars, inflammation, Kaposi's sarcoma, liver cirrhosis, benign neoplasia (e.g., hemangiomas), malignant neoplasia (e.g., skin cancer, cutaneous malignancies, and other malignancies), onchocerciasis, psoriasis, growth of solid tumors, metastatic spread of solid tumors, and warts.
  • the anti-Ov-ASP factor may be administered in an amount which is effective to inhibit angiogenesis n the subject. This amount may be readily determined by the skilled artisan.
  • the present invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising an anti-Ov-ASP factor and a pharmaceutically-acceptable carrier.
  • the pharmaceutically-acceptable carrier must be “acceptable” in the sense of being compatible with the other ingredients of the composition, and not deleterious to the recipient thereof.
  • acceptable pharmaceutical carriers include carboxymethyl cellulose, crystalline cellulose, glycerin, gum arabic, lactose, magnesium stearate, methyl cellulose, powders, saline, sodium alginate, sucrose, starch, talc, and water, among others.
  • Formulations of the pharmaceutical composition may conveniently be presented in unit dosage. The formulations may be prepared by methods well-known in the pharmaceutical art.
  • the active compound may be brought into association with a carrier or diluent, as a suspension or solution.
  • a carrier or diluent as a suspension or solution.
  • one or more accessory ingredients e.g., buffers, flavoring agents, surface active agents, and the like
  • the choice of carrier will depend upon the route of administration.
  • the pharmaceutical composition would be useful for administering the anti-Ov-ASP factor to a subject to treat a disease or condition where the inhibition of angiogenesis would be desirable, including, without limitation, benign neoplasia, malignant neoplasia, and onchocerciasis.
  • the anti-Ov-ASP factor is provided in an amount which is effective to inhibit angiogenesis in the subject. This amount may be readily determined by the skilled artisan.
  • O. volvulus O. volvulus worms in the eye.
  • This response is initially manifested in the development of new blood vessels (neovascularization), and later in the opacification of the cornea.
  • Animal models have shown that development of O. volvulus -mediated corneal inflammation (keratitis) results from the temporal recruitment to the cornea of neutrophils and eosinophils, through the network of new blood vessels.
  • the inflammatory process is dependent on development of a systemic T helper type 2 (Th2)-type response to the parasite antigens [1-4].
  • Th2 systemic T helper type 2
  • Vespid venoms (bee and hornet venoms) are important allergens of humans. Furthermore, vespid venom antigen 5 has similarities to the testis-specific protein (Tpx)/cysteine-rich secreted protein (CRISP) families of proteins [8]. These families of proteins include a major autoantigen of the mammalian sperm acrosome, and are found in many other vertebrate tissues [8, 9].
  • Tpx testis-specific protein
  • CRISP cysteine-rich secreted protein
  • Ov-ASPs are similar to a component of vespid venoms, and vespid venoms are capable of inducing both allergic and inflammatory responses [10], it was hypothesized that these proteins may play a role in the development of ocular onchocerciasis.
  • the inventors present data demonstrating that two Ov-ASP proteins induce an angiogenic response in naive mouse corneas, thereby suggesting that these proteins may play a direct role in the pathogenesis of ocular onchocerciasis, possibly by promoting neovascularization during nodule formation.
  • the inventors isolated a cDNA encoding one member of the Ov-ASP protein family by immunoscreening an L3 cDNA expression library (OvB93-RP, GenBank accession number AF020586).
  • the isolated cDNA was designated Ov-asp-1.
  • the complete nucleotide sequences of two other members of the Ov-asp family were identified as part of the O. volvulus EST sequencing project, and have been designated Ov-asp- 2 (GenBank accession number H39490) and Ov-asp-3 (GenBank accession number AA917267).
  • the present inventors designed three sets of primers flanking the complete open reading frames of each of the clones with EcoR1 sites at their 5′ ends to facilitate subsequent cloning.
  • the nucleotide sequences of the primers were as follows:
  • Ov-asp-1 exp-r 5′-TCATTTTCTGCACAGTCCAGA-3′ (SEQ ID NO:9)
  • gene-specific primers were designed to amplify an approximately 400-bp fragment of the 3′ region of each of the cDNA clones. Amplifications were also carried out employing primers for a cDNA encoding the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (Ov-gpd-1) as an internal control.
  • the gene-specific primers used were:
  • Ov-asp-1-f 5′-AAAACTGCTGGTACGGA-3′ (SEQ ID NO:14)
  • Ov-asp-2-f 5′-GTTTACACCCAGCGGTCAGATAC-3′ (SEQ ID NO:16)
  • Ov-asp-3-f 5′-CGCCTTAGGGTTACCAAAAGATG-3′ (SEQ ID NO:18)
  • Ov-asp-3-r 5′-CAAAAATTAAATGAAGTGAAACG-3′ (SEQ ID NO:19)
  • Ov-gpd-1-f 5′-TGATCTCACTTGCCGACTGC-3′ (SEQ ID NO:20)
  • Ov-gpd-1-r 5′-AAGGTGTTGTCAGAAGGC-3′ (SEQ ID NO:21)
  • PCR products were analyzed by agarose gel electrophoresis.
  • Southern blots were prepared from BamHl and HindIII restricted human O. volvulus genomic DNA samples, as previously described [11]. The blot was sequentially probed with purified insert DNA from a clone encoding the Ov-asp-2 open reading frame, then with a purified DNA fragment encoding a portion of O. volvulus prolyl 4-hydroxylase. The blot probed with the Ov-asp-2 insert was washed under conditions of moderate stringency (0.4 ⁇ SSC, 1% SDS, at 48° C.), while the blot hybridized with the O. volvulus prolyl 4-hydroxylase insert was washed at high stringency (0.2 ⁇ SSC, 1% SDS, at 65° C.).
  • cDNAs encoding the three members of the Ov-ASP family, were isolated by PCR amplification from a cDNA library prepared from O. volvulus infective larvae, as described in Materials and Methods.
  • the derived amino acid sequences of the three members of the Ov-ASP family are presented in FIG. 1 . All three proteins have characteristics similar to members of the Tpx and CRISP protein families.
  • the Ov-ASPs are 54-62% identical to each other, and contain 6 of the 10 conserved cysteine residues found in the vertebrate members of the Tpx family of proteins. All three of the members of the Ov-ASP family contain putative signal sequences at their amino terminal ends.
  • the three Ov-ASPs contain the sequence HFTQ, or a closely related variant (NFTQ), which is a conserved variation of the HYTQ sequence found in most members of the CRISP family [7].
  • O. volvulus clones encoding homologues of the vespid antigens suggest that the Ov-asp clones are members of a multigene family.
  • a Southern blot prepared from O. volvulus genomic DNA was probed under conditions of moderate stringency with a purified insert encoding the Ov-asp-2 open reading frame.
  • the Ov-asp-2 cDNA hybridized to a large number of DNA bands in both HindlIl and BamHl restricted O. volvulus DNA, ranging in size from 3.5 to 16 kbp (FIG. 3 ). In contrast, no hybridization to human genomic DNA was detected (FIG. 3 ).
  • volvulus EST database (as of 1999), using gene-specific segments of the three Ov-asp cDNAs, showed that the Ov-asp-3 is L3-specific, while the Ov-asp-1 and the Ov-asp-2 are both present in the mL3, and up-regulated in the L3 stage (Table 1).
  • the Ov-asp gene cluster comprises up to 0.81% of the L3/mL3 ESTs in the current O. volvulus EST database.
  • Transcripts of Ov-asp-1 and Ov-asp-2 could be amplified from other life stages of the parasite, but no corresponding ESTs were identified in the present EST data sets of any other stage (mf, L2, male, female). Taking into account the relatively smaller number of ESTs in these data sets, these results suggest that the Ov-asp-1 and Ov-asp-2 transcripts are probably down-regulated in these stages.
  • Ov-ASP-1 and Ov-ASP-2 were expressed in E. coli as soluble fusion proteins with maltose-binding protein (MBP), and purified to homogeneity by amylose affinity chromatography. Thirty ⁇ g of each purified protein were injected into the corneas of naive BALB/c mice, and growth of blood vessels was monitored by slit-lamp examination. As shown in FIG.
  • the Ov-ASP-2/MBP construct induced a pronounced angiogenic response, with vessels growing from the peripheral cornea to the site of injection. Vessel length was maintained throughout the 7-day period, although the area of the cornea in which vessels were present (measured as clock hours) decreased after 5 days. Similar kinetics were found after injection of 10 ⁇ g of protein (data not shown).
  • mice were injected with Ov-ASP-1/MBP, Ov-ASP-2/MBP, MBP alone, or with O. volvulus protein disulfide isomerase fused with MBP (Ov-PDI/MBP) [18].
  • Ov-PDI/MBP O. volvulus protein disulfide isomerase fused with MBP
  • mice injected with Ov-ASP-2/MBP or the control proteins were removed, then fixed in formalin. Paraffin sections (5 pm) were examined after staining with hematoxylin and eosin. Corneas of Ov-ASP-2/MBP-injected animals had numerous blood vessels, but relatively few inflammatory cells (FIG. 7 ). In contrast to the model of O. volvulus keratitis in which there is an intense neutrophilic and eosinophilic infiltrate [1], most of the inflammatory cells in Ov-ASP-2/MBP-injected corneas were mononuclear. Similar results were seen after injection of Ov-ASP-1/MBP, whereas no vessels or inflammatory cells were detected in the corneas of animals injected with Ov-PDI/MBP or MBP alone (data not shown).
  • Ov-ASP-1/MBP and Ov-ASP-2/MBP also differed significantly from that seen in animals immunized subcutaneously and challenged intrastromally with soluble native parasite antigens—a procedure that induces severe corneal opacification [3, 18].
  • Ov-ASP proteins may directly induce an angiogenic response and may therefore contribute to corneal neovascularization in onchocercal keratit is.
  • corneal neovascularization is important in the development of ocular pathology associated with O. volvulus infection, angiogenesis may also be essential for maintaining the nodule in which the adults reside.
  • These nodules are highly vascularized [20]; their survival may depend on production of angiogenic proteins in a manner similar to that described for tumors [21]. It may be that one function of the members of the Ov-ASP family is to promote neovascularization during nodule formation, thereby ensuring a sufficient blood supply to the adult parasite.
  • Kenyon et al. A model of angiogenesis in the mouse cornea. Inv. Ophthamol. Vis. Sci ., 37:1625-32, 1996.

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WO2017165420A1 (fr) * 2016-03-21 2017-09-28 Hawdon John M Ankylostomes humains modifiés utilisés comme nouveau système de bioadministration pour vaccins et substances biologiques

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Cited By (9)

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WO2005122739A3 (fr) * 2004-06-15 2006-08-10 New York Blood Ct Inc Propriete d'adjuvance et de stimulation des fonctions immunitaires de produits naturels de onchocerca volvulus
JP2008502695A (ja) * 2004-06-15 2008-01-31 ザ ニューヨーク ブラッド センター インコーポレイテッド 回旋糸状虫の天然産物のアジュバント性及び免疫増強特性
US20090191229A1 (en) * 2004-06-15 2009-07-30 New York Blood Center Adjuvancy and immune potentiating properties of natural products of onchocerca volvulus
US7700120B2 (en) 2004-06-15 2010-04-20 New York Blood Center Adjuvancy and immune potentiating properties of natural products of Onchocerca volvulus
US20100266634A1 (en) * 2004-06-15 2010-10-21 New York Blood Center, Inc. Adjuvancy and immune potentiating properties of natural products of onchocerca volvulus
JP4825797B2 (ja) * 2004-06-15 2011-11-30 ニューヨーク・ブラッド・センター・インコーポレーテッド 回旋糸状虫の天然産物のアジュバント性及び免疫増強特性
US9017699B2 (en) 2004-06-15 2015-04-28 New York Blood Center, Inc. Adjuvancy and immune potentiating properties of natural products of Onchocerca volvulus
US9255140B2 (en) 2004-06-15 2016-02-09 New York Blood Center, Inc. Adjuvancy and immune potentiating properties of natural products of Onchocerca volvulus
WO2017165420A1 (fr) * 2016-03-21 2017-09-28 Hawdon John M Ankylostomes humains modifiés utilisés comme nouveau système de bioadministration pour vaccins et substances biologiques

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